Gene responsible for stargardt-like dominant macular dystrophy

ABSTRACT

The gene responsible for Stargardt-like macular dystrophy has been identified, along with its normal allelic form. The mutant gene encodes a mutant protein containing a frameshift mutation, resulting in abnormal fatty acid synthesis and transport in the retina. Also disclosed are assays for Stargardt-like macular dystrophy and therapies.

FIELD OF THE INVENTION

[0001] This invention relates to the gene responsible for causingStargardt-like dominant macular dystrophy, and to assays which use thisgene or the protein encoded by it, and to methods of treating thiscondition by administering the protein.

BACKGROUND

[0002] Macular dystrophy is a term applied to a heterogeneous group ofdiseases that collectively are the cause of severe visual loss in alarge number of people. A common characteristic of macular dystrophy isa progressive loss of central vision resulting from the degeneration ofphotoreceptor cells in the retinal macula. In many forms of maculardystrophy, the end stage of the disease results in legal blindness. Morethan 20 types of macular dystrophy are known: e.g., age-related maculardystrophy, Stargardt-like dominant macular dystrophy, recessiveStargardt's disease, atypical vitelliform macular dystrophy (VMD1),Usher Syndrome Type 1B, autosomal dominant neovascular inflammatoryvitreoretinopathy, familial exudative vitreoretinopathy, and Best'smacular dystrophy (also known as hereditary macular dystrophy or Best'svitelliform macular dystrophy (VMD2). For a review of the maculardystrophies, see Sullivan & Daiger, 1996, Mol. Med. Today 2:380-386.

[0003] Stargardt-like dominant macular dystrophy (also called autosomaldominant macular atrophy) is a juvenile-onset macular degeneration.Patients afflicted with this disease generally have normal vision asyoung children, but during childhood, visual loss begins, which rapidlyprogresses to legal blindness. Clinically it is characterized by thepresence of an atrophic macular lesion with sharp borders and is oftenassociated with yellow fundus flecks. The pathological features seen inStargardt-like dominant macular dystrophy are in many ways similar tothe features seen in age-related macular dystrophy (AMD), the leadingcause of blindness in older patients in the developed world.

[0004] AMD is an extraordinarily difficult disease to study genetically,since by the time patients are diagnosed, their parents are usually nolonger living and their children are still asymptomatic. Thus, familystudies which have led fo the discovery of the genetic basis of manyother diseases have not been practical for age-related maculardystrophy. As there are currently no widely effective treatments forAMD, it is hoped that study of Stargardt-like dominant maculardystrophy, and in particular the discovery of the underlying geneticcause of Stargardt-like dominant macular dystrophy, will shed light onage-related macular dystrophy as well. A significant proportion of theAMD cases is caused by recessive mutations in the recessive Stargardtdisease gene. (Allikmets, et al 1997 Science 277:1805-1807).

[0005] It seems reasonable to suggest that mutations within the diseasegene responsible for Stargardt-like dominant macular dystrophy whichclosely resembles the recessive Stargardt disease may be responsible forthe significant proportion of AMD cases. It would be desirable tocharacterize the gene responsible for this disease in order to have abetter understanding of this disease and to elucidate its potential rolein other forms of macular degeneration.

DETAILED DESCRIPTION OF THE INVENTION

[0006] In accordance with this invention, a mutant gene responsible forautosomal dominant Stargardt-like macular dystrophy has been identifiedand sequenced. Additionally, the normal allelic form of this gene hasalso been identified and sequenced.

[0007] A new gene, presently designated “ELF” (for Elongation of FattyAcids), is potentially involved in the elongation pathway for thesynthesis of decosahexaenoic fatty acid (DHA), a critical component inretinas. The mutant version of this gene contains a 5-base pair deletionwhich causes a frameshift mutation. The resultant mutated protein doesnot function in the DHA pathway, resulting in retinal dysfunction.

[0008] Thus one aspect of this invention is a nucleic acid encoding thenormal form of ELF protein, which is free from associated nucleic acids.In preferred embodiments, the nucleic acid sequence is a DNA, and inmore preferred embodiments it is a cDNA.

[0009] Another aspect of this invention is a nucleic acid encoding amutant form of ELF, which is free from associated nucleic acids. Inpreferred embodiments, the nucleic acid is a DNA, and in more preferredembodiments, it is a cDNA.

[0010] Another aspect of this invention are the novel proteins, normalELF and its mutant form, free from associated proteins. Also part ofthis invention are fragments of these proteins which retain at least onebiological activity.

[0011] A further aspect for this invention is a method of treatingindividuals who suffer from Stargardt-like macular dystrophy comprisingadministering to the individual an effective amount of ELF protein. TheELF protein may be in a pharmaceutically acceptable carrier, and it maybe administered in the form of eyedrops or other ophthalmic preparation.

[0012] Another aspect of this invention is a method of treatingindividuals who suffer from Stargardt-like macular dystrophy comprisingintroducing a nucleic acid encoding the ELF protein into the individual.This gene therapy approach may involve the use of viral vectors, such asadenovirus, or it may involve the use of plasmid DNA.

[0013] Yet another aspect of this invention are assays to identify if anindividual is at risk for Stargardt-like macular dystrophy comprisingdetermining if the individual's DNA contains a gene for a mutant form ofELF.

[0014] Another aspect of this invention is the use of ELF gene's 5′regulatory region for targeting the expression of genes specifically tophotoreceptor cells of the retina for gene therapy of maculardegeneration.

[0015] Another aspect of this invention is the use of mouse ELF DNA ormouse ELF protein corresponding to the normal or mutant form of humanELF for generating an animal model (knock-out or transgenic) that can beused for testing the anti-AMD compounds.

[0016] A further aspect of this invention are methods of producing longchain fatty acids using DNA encoding ELF or using ELF protein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 is the protein for normal human ELF protein (SEQ.ID.NO. 1).The underlined amino acids represent 5 putative transmembrane segments.The HXXHH motif between predicted membrane spanning regions 2 and 3 thatis characteristic of dioxy iron cluster proteins is double underlined.The protein fragment deleted in patients with Stargardt-like maculardystrophy is shown in italics. The cytosolic carboxy-terminal dilysinemotif responsible for the retrieval of trans-membrane proteins fromcis-Golgi to the endoplasmic reticulum is shown in bold italics.

[0018]FIG. 2 is the protein for mutant human ELF protein which causesStargardt-like dominant macular dystrophy (SEQ.ID.NO. 2). Underlinedamino acids represent four of five putative transmembrane segments; thefragment of the fifth transmembrane segment that is common for normaland mutant alleles of the protein is highlighted by the dotted line. TheHXXHH motif between predicted membrane spanning regions 2 and 3 that ischaracteristic of dioxy iron cluster proteins is double underlined. Theprotein fragment generated by the 5-bp deletion in patients withStargardt-like macular dystrophy is shown in italics.

[0019]FIG. 3 is normal human ELF cDNA (SEQ.ID.NO. 3) and the amino acidsequence (SEQ.ID.NO. 1) of the human ELF protein. Underlined nucleotidesin bold encompassing base pairs 797-801 represent the deletion found inpatients with dominant Stargardt-like macular dystrophy. The proteinfragment deleted in patients with Stargardt-like macular dystrophy isshown in bold underline

[0020]FIG. 4 is mutant human ELF cDNA (SEQ.ID.NO. 4) and the amino acidsequence (SEQ.ID.NO. 2) of the human mutant ELF protein. The region ofthe protein encompassing amino acids 264-271 (bold underlined) representa fragment generated as a result of the 5-base pair deletion in patientswith dominant Stargardt-like macular dystrophy.

[0021]FIG. 5 is the protein for normal mouse ELF protein (SEQ.ID.NO. 5).Underlined amino acids represent 5 putative transmembrane segments. TheHXXHH motif between predicted membrane spanning regions 2 and 3 that ischaracteristic of dioxy iron cluster proteins is double underlined. Theprotein fragment similar to the human ELF fragment deleted in patientswith Stargardt-like macular dystrophy is shown in italics. Cytosoliccarboxy-terminal dilysine motif responsible for the retrieval oftransmembrane proteins from cis-Golgi to the endoplasmic reticulum isshown in bold italics.

[0022]FIG. 6 is mouse cDNA for ELF (SEQ.ID.NOS. 6) and the amino acidsequence (SEQ.ID.NO.: 5) of the mouse ELF protein.

[0023]FIG. 7 shows the genomic DNA sequence of the ELF gene (SEQ.ID.NO.:8). Underlined nucleotides in capitals represent exons. Initiating ATGcodon in exon 1 and terminating TAA codon in exon 6 are shown in bolditalics. The exact lengths of the gaps between the exons are unknown;these gaps are presented as runs of ten bold n as a convenience only.

[0024]FIG. 8 shows the pairwise comparison of human and mouse ELFproteins. The upper amino acid sequence shown is the human ELF protein(SEQ.ID.NO. 1). The lower amino acid sequence shown is the mouse ELFprotein (SEQ.ID.NO. 5). The two proteins are highly identical whichindicates they are true orthologues. Both proteins share the cytosoliccarboxy-terminal dilysine motif responsible for the retrieval oftransmembrane proteins from cis-Golgi to the endoplasmic reticulum (twolysines are located at −3 and −5 positions with respect to the carboxylterminus).

[0025]FIG. 9 depicts the sequence alignment of the human ELF protein(SEQ.ID.NO. 1) and its two yeast homologues, Elo2p (SEQ.ID.NO. 8) andElo3p (SEQ.ID.NO. 9) from Oh et al., 1997 J. Biol. Chem 272:17376-17384.The degree of homology is high enough to assign the function ofelongation of fatty acids to the human ELF protein.

[0026]FIG. 10 depicts the enzymatic conversions involved in the linoleicacid (n−3) and alpha-linolenic acid (n−6) pathways of essential fattyacid synthesis, including three elongation steps required of thebiosynthesis of DHA

[0027]FIG. 11 shows a Kyte-Doolittle hydropathy plot of human ELF.Numbers 1 to 5 mark putative transmembrane segments. The hydropathy plotand membrane topology of human ELF (SEQ.ID.NO. 1)are similar to thoseproposed for its two yeast homologues, Elo2p (SEQ.ID.NO. 8) and Elo3p(SEQ.ID.NO. 9), experimentally shown to be involved in elongation offatty acids.

[0028]FIG. 12 shows association (segregation) of the 5 base pairdeletion within the ELF gene with the disease phenotype in the familywith dominant Stargardt-like macular dystrophy. The figure shows thestructure of this pedigree and four sequencing runs (boxed) of PCRfragments that represent exon 6 and adjacent intronic regions of thehuman ELF gene (SEQ.ID.NOS.: 10, 11, 12, and 13). From left to right,the runs are from A40 (father, unaffected with Stargardt-like dominantmacular dystrophy), A4 (mother, affected with Stargardt-like dominantmacular dystrophy), A430 (son of A4 and A40, unaffected withStargardt-like dominant macular dystrophy), A43 (daughter of A4 and A40,affected with Stargardt-like dominant macular dystrophy). Reading theboxed chromatograms from left to right, the 5-base pair deletion showsup as appearance of double peaks starting from position 7 in the case ofpatients A4 and A43.

[0029]FIGS. 13A and 13B show the result of in situ hybridization of thehuman ELF mRNA in rhesus monkey retina. FIG. 13A shows specificexpression in the inner segments of photoreceptor cells with theantisense probe. Probe signal is indicated by the arrow; retinal layersare visualized with propidium iodide counterstain. FIG. 13B shows thehybridization with the sense control probe (sense probe is notcomplementary to the ELF mRNA). Retinal layers are marked as RPE,retinal pigment epithelium; OS, outer segments of photoreceptors; IS,inner segments of photoreceptors; ONL, outer nuclear layer; OPL, outerplexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer;GCL, ganglion cell layer. PhumGL1/HR2 is a hybridization probe thatrepresents a fragment of the human normal ELF cDNA (SEQ.ID.NO. 4). withcoordinates 561-771.

[0030]FIG. 14 shows the expression pattern of the ELF gene in 10 humantissue plus retinal pigment epithelium-derived cell line ARPE19, asdetermined by RT-PCR amplification with 17 cycles. The expression isdetected in human retina only.

[0031]FIG. 15 shows the expression pattern of the human ELF gene inhuman tissues as determined by Northern blot hybridization. Theexpression is prominent in the human retina; the hybridization signal isalso seen in the human brain. ELF mRNA exists in two different species,similar to what was reported for its only mammal relative, the Cig30gene (Tvrdik et al 1999, J. Biol. Chem. 274:26387-26392).

[0032]FIG. 16 shows the 5′-regulatory region of human ELF gene. Theinitiating ATG codon in the first exon is shown in bold. Sequenceelements that are common to mammalian RNA polymerase II promoters (CAATbox at position 1657 and four GC boxes at positions 1446, 1513, 1585,and 1744) are shown in bold and underlined.

[0033] As Used Through the Specification and Claims, the FollowingDefinitions Apply:

[0034] “Free from associated nucleic acids” means the nucleic acid isnot covalently linked to a nucleic acid which is naturally linked to inan individual's chromosome.

[0035] “Free from associated proteins” means the ELF protein is notlocated in its native cell membrane; or in the case of the mutantallele, the mutant ELF is not located in the retinal cytoplasm where itnormally is found.

[0036] This invention relates to the identification and characterizationof the mutant allele responsible for Stargardt-like macular dystrophy.The designation of the gene is EFL (for Elongation of Fatty Acids).

[0037] Essential fatty acids (EFAs) are polyunsaturated fatty acids thatcannot be manufactured by mammals, yet are required for a number ofimportant biochemical processes, and thus must be supplied in the diet.The most important dietary EFAs are linoleic acid and alpha-linolenicacid (ALA). These two EFAs undergo a number of biosynthetic reactionsthat convert them into various other EFAs. FIG. 10 depicts thebiosynthetic reactions involving the two groups of EFAs, the n−6 EFAs(linoleic acid derivatives) and the n−3 EFAs (ALA derivatives). EFAs areformed from linoleic acid and ALA by a series of alternating reactionsinvolving the removal of two hydrogens coupled with the insertion of anadditional double bond (desaturation) and the lengthening of the fattyacid chain by the addition of two carbons (chain elongation). The endproduct of the ALA pathway is docosahexaenoic acid (DHA).

[0038] Decosahexaenoic fatty acid (DHA) is a highly polyunsaturated,long-chain fatty acid, which has six double bonds and is 22 carbons inlength [indicated as 22:6 (n−3), where the first number indicates chainlength, the second number indicates the number of double bonds, and“n−3” indicates the position of the first double bond as its relates tothe terminal methyl group]. DHA is a critical component of membranes invertebrate retina, comprising up to 50% of all fatty acids inphotoreceptor cells. While not wishing to be bound by theory, it appearsthe normal allele of ELF is involved in one of the elongation stepsduring DHA synthesis.

[0039] Bioinformatic analysis revealed a weak but significant homologybetween ELF and a group of two yeast proteins (Elo2p and Elo3p), whosefunction are also the elongation of fatty acids. The Kyte-Doolittlealgorithm (FIG. 11) predicts that ELF has a transmembrane organizationinvolving five transmembrane regions which is similar to the reportedtransmembrane organization of Elo2p and Elo3p. The Elo2p and Elo3pproteins are necessary for the synthesis of very long chain fatty acidsof up to 24 and 26 carbon atoms, respectively (Oh et al. 1997, J. Biol.Chem. 272:17376-17384, which is hereby incorporated by reference). Itseems that human ELF protein is responsible for the biosynthesis of DHA,as it requires the elongation up to 24 carbon atoms with subsequentchain shortening (beta-peroxidation) to 22 carbon atoms.

[0040] The mutant (i.e. disease-causing allele) of ELF contains a 5 bpdeletion starting at bp 797. This results in a frameshift mutation fromthis position through the remainder of the C-terminus. The mutationremoves the C-terminal region of the ELF protein which is reasonablyconserved between human and mouse (see FIG. 8). Evolutionaryconservation indicates functional significance of the protein regionremoved as a result of the frameshift mutation. In addition, theframeshift mutation removes the targeting signal in the C-terminus whichis the same sequence as those known to be responsible for targetingproteins to the endoplasmic reticulum (Gaynor et al. 1994 J. Cell Biol.127:653-665 and Schroder et al. 1995 J. Cell Biol. 131:895-912, both ofwhich are incorporated by reference). This would prevent ELF proteinfrom trafficking to the site of biosynthesis of very long chain fattyacids (membranes of the endoplasmic reticulum) Thus, deficiencies in thebiosynthesis of DHA or other retina-specific fatty acids with very longchain resulting from mutations in ELF would predictably lead to retinaldysfunction.

[0041] There are additional observations which indicate that the genesof this invention are involved in Stargardt-like macular dystrophy.First of all, the mutant (disease-causing allelic form) has beenidentified in three independent families with Stargardt-like maculardystrophy. Secondly, the gene maps to the genetically defined region onhuman chromosome 6q14, which has been identified with Stargardt-likemacular dystrophy. The ELF gene maps to the PAC clone dJ94c4 which islocated in close vicinity of the genetic marker D6S460. The maximumreported lod score for D6S460 was 9.3, which is a clear indication ofgenetic proximity of this marker to the disease locus (Edwards et al.1999, Am. J. Ophthal. 127:426435.) Further, this gene was found to beexclusively expressed in the retina, specifically, in the photoreceptorcells (see FIGS. 13, 14, and 15).

[0042] Nucleic Acids

[0043] Thus, one aspect of this invention are nucleic acids which encodeeither the normal allele or the mutant allele of ELF; these nucleicacids may be free from associated nucleic acids. Preferably the sourceof the nucleic acids is a human; although this invention includes othermammalian forms, such as mouse, rat, pig, monkey and rabbit. Genesencoding ELF from a non-human mammal can be obtained by using the humanDNA as a probe in libraries of the retina using standardbiotechnological techniques, and one aspect of this invention is amethod of isolating a non-human nucleic acid encoding an ELF proteincomprising probing a retinal library of a non-human mammal. The probe ispreferably from the human or mouse DNA,

[0044] As used throughout the specification and claims, the term “gene”specifically refers to the protein-encoding portion of the gene, i.e.the structural gene, and specifically does not include regulatoryelements such as promoters, enhancers, transcription termination regionsand the like. The gene may be a cDNA or it may be an isolated form ofgenomic DNA. As used herein, “isolated” means that the DNA is physicallyseparated from the DNA which it is normally covalently attached to inthe chromosome. This includes DNA with a heterologous promoter and DNAwhich has its native regulatory sequences, but is not present in itsnative chromosome.

[0045] The ELF genes of this invention (both allelic forms) may havetheir own regulatory sequences operatively linked, or one may, usingknown biotechnology techniques, operatively linked heterologousregulatory regions. Such regulatory regions are well known, and includesuch promoters as the CMV promoter, rod-specific promoter of therodopsin gene, retinal pigment epithelium-specific promoters ofbestrophin or RPE65 genes. Commercially available mammalian expressionvectors which are suitable for the expression of human ELF DNA include,but are not limited to: pMC1neo (Stratagene), pSG5 (Stratagene), pcDNAIand pcDNAIamp, pcDNA3, pcDNA3.1, pCR3.1 (Invitrogen), EBO-pSV2-neo (ATCC37593), pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224),pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), and pSV2-dhfr (ATCC 37146).

[0046] The ELF genes (regardless of species and allelic form) andoperatively linked regulatory regions (an “ELF expression cassette) maybe placed in a vector for transfer into a host cell. Vectors which arepreferred include plasmids and, to a lesser degree, viral vectors. Thechoice of vector will often be dependent upon the host cell chosen.Cells which are preferred host cells include but are not limited to:ARPE-19, RPE-J, Y79, L cells L-M(TK⁻) (ATCC CCL 1.3), L cells L-M (ATCCCCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70),COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3(ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C1271 (ATCCCRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).

[0047] A further aspect of this invention is a method of making an ELFprotein (either a mutant or normal allelic form) comprising culturing ahost cell comprising an ELF expression cassette, and recovering ELFprotein. Alternatively a ELF gene may be integrated into a chromosome ofthe host cell, rather than being located on a vector. The resultantELF-expressing cell lines (comprising a heterologous ELF gene, whetheron a vector or in a host's chromosome) make up yet another aspect ofthis invention.

[0048] ELF Protein

[0049] Another aspect for this invention is an allelic form of ELFprotein (normal or mutant) which is free from associated proteins. In apreferred embodiment the protein is mammalian, and in more preferredembodiments, the protein is a human form.

[0050] Still another aspect of this invention is a method for treating,preventing or lessening the severity of Stargardt-like maculardegeneration comprising administering the normal allelic form of ELF toan individual at risk of the disease or who manifests the symptoms ofthe disease. The normal allelic form of ELF is preferably recombinantlyproduced. The normal ELF can substitute for the defective ELF made bythese individuals, and perform the normal transporting function. Theadministration of the ELF protein is preferably in the form of apharmaceutical composition comprising pharmaceutically acceptablediluents, excipients, and optionally stabilizers or preservatives. Atypical pharmaceutical composition comprises 0.1 to 95% protein and isadministered once, twice or three times daily. The pharmaceuticalcomposition is preferably in the form of eyedrops, solutions orsuspensions for subretinal and intravitreal injections, or slow releasepellets.

[0051] Still another aspect of this invention is a method for in vitrobio-synthesis of fatty acids with a very long chain, for example DHA.Biosynthesis of DHA involves several elongation and desaturation steps(see FIG. 10).

[0052] We have previously identified and patented a retina-specificdelta 6 desaturase called CYB5RP (U.S. Provisional Application SerialNo. 60/103,760; PCT/US99/23253, which is hereby incorporated byreference). CYB5RP is homologous to a delta 6 desaturase from Boragooficinalis. Both CYB5RP and this Borago delta 6 desaturase, unlikedesaturases from higher plants, are unusual in containing a cytochromeb5-like domain fused to their N-termini (Sayanova et al., 1997, Proc.Natl. Acad. Sci. USA 94:4211-4216; hereinafter “Sayanova”, which ishereby incorporated by reference). The Borago desaturase has beenexpressed in transgenic tobacco, resulting in high levels of delta 6desaturated fatty acids in the transgenic tobacco leaves, including highlevels of γ-linolenic acid (GLA) (Sayanova). Similarly, CYB5RP,expressed in transgenic plants (e.g., tobacco) is expected to provide avaluable source of GLA. Co-expression of the ELF cDNA in the same plantwould predictably couple elongation and desaturation steps required forthe production of DHA. Thus, CYB5RP and ELF DNA, co-expressed intransgenic plants, is expected to provide a valuable source of theimportant nutrient-docosahexaenoeic acid (DHA). The protocols forexpression of foreign genes in plants are well developed and reported inthe literature (Sayanova).

[0053] Animal Model

[0054] Another aspect of this invention is the use of mouse ELF DNA ormouse ELF protein corresponding to the normal or mutant form of humanELF for generating an animal model (knock-out or transgenic) that can beused for testing anti-AMD compounds. Oligonucleotide primers designedfrom the mouse cDNA sequence (SEQ.ID.NOS. 6) can be used to PCR amplifya fragment of the mouse ELF gene from the DNA of 129-strain embryonicstem cells (DNA of the 129Sv/J lambda genomic library is available fromStratagen). This genomic fragment can be used to generate a constructthat will, upon electroporation into the 129-strain ES cells, generate anull mutation (targeted disruption) of the ELF gene. ES clones that haveundergone homologous recombination with the construct can be injectedinto C57BL/6 blastocysts. Injected blastocytes can be transplanted intothe uterus of pseudopregnant female mice. Their progeny can be selectedfor the germine transmission of the disrupted ELF gene and bred with129SVEV females. The animals with heterozygous disruption of the mouseELF gene can be bred to homozygosity.

[0055] The art of constructing the knock-out and transgenic mouse modelsis well-described in the literature and exemplified in Weng et al., 1999Cell 98:13-23, which is hereby incorporated by reference.

[0056] Assays for Mutant Forms

[0057] Another aspect of this invention is an assay to identifyindividuals who are at risk for developing the symptoms ofStargardt-like macular dystrophy. The children of a person who has thisdisease are at risk, as the disease is inherited in a dominant-Mendelianfashion. Thus, if one parent does not have the disease, and the secondparent is a heterozygous afflicted patient, the children have a 50%probability of developing the disease. As the children begin life withnormal eyesight, there is time to intervene with protein therapy toreduce the severity, delay onset, or even completely prevent thesymptoms from developing.

[0058] One assay in accordance with this invention is a labeled nucleicacid probe which spans the portion of the nucleic acid just 5′ to thearea where the mutant deletion occurs, and includes base pairs after thedeletion, which include the frameshift mutation. Referring to the normalallele (SEQ.ID.NO. 3), a probe would be of any convenient length,preferably about 15 to 35 bp in length, more preferably at least about25-30 base pairs in length. It would include a desired number of basepairs up to 796, skip 797-801, and resume at 802. The probe can beconstructed so that it would hybridize to the sense strand, oralternatively so that hybridization occurs with the anti-sense strand. Atypical probe would thus comprise (where the superscripted numeralcorrespond to base pair positions according to the normal allele): C⁷⁹⁰T⁷⁹¹ T⁷⁹² T⁷⁹³ C⁷⁹⁴ T⁷⁹⁵ T⁷⁹⁶ C⁸⁰² T⁸⁰³ A⁸⁰⁴ C⁸⁰⁵ A⁸⁰⁶ T⁸⁰⁷ T⁸⁰⁸ C⁸⁰⁹(SEQ.ID.NO. 14). The probe may contain additional 5′ and or 3′-terminusbase pairs which are essentially identical to those in the normalallele, so that the length of the probe is at least 15 bp long, andpreferably at least 25 bp long.

[0059] Generally the probe includes a detection means, such as adetectable label. Such labels, including radiolabels or fluorescentlabels are well known in the art.

[0060] In an alternative embodiment, the probe would include base pairswhich would hybridize to the normal allelic form of the ELF gene, butwould not hybridize to the mutant form.

[0061] Another embodiment of this invention is a method of determiningif an individual is at risk of developing Stargardt-like maculardystrophy comprising obtaining a sample of the ELF protein produced bythe individual, and determining whether it is the normal or mutant form.This is preferably done by determining if an antibody specific for thenormal allele of the ELF protein binds to the protein produced by theindividual. In an alternate embodiment of this assay, the antibody isspecific for the mutant form of ELF.

[0062] The antibodies of these assays may be polyclonal antibodies ormonoclonal antibodies. The antibodies can be raised against theC-terminal peptide which is different in normal and mutant ELF proteins.The antibodies can be raised against the allele-specific syntheticC-terminal peptides that are coupled to suitable carriers, e.g., serumalbumin or keyhole limpet hemocyanin, by methods well known in the art.Methods of identifying suitable antigenic fragments of a protein areknown in the art. See, e.g., Hopp & Woods, 1981, Proc. Natl. Acad. Sci.78:3824-3828; and Jameson & Wolf, 1988, CABIOS (Computer Applications inthe Biosciences) 4:181-186, both of which are hereby incorporated byreference.

[0063] For the production of polyclonal antibodies, ELF protein or anantigenic fragment, coupled to a suitable carrier, is injected on aperiodic basis into an appropriate non-human host animal such as, e.g.,rabbits, sheep, goats, rats, mice. The animals are bled periodically andsera obtained are tested for the presence of antibodies to the injectedantigen. The injections can be intramuscular, intraperitoneal,subcutaneous, and the like, and can be accompanied with adjuvant.

[0064] For the production of monoclonal antibodies, ELF protein or anantigenic fragment, coupled to a suitable carrier, is injected into anappropriate non-human host animal as above for the production ofpolyclonal antibodies. In the case of monoclonal antibodies, the animalis generally a mouse. The animal's spleen cells are then immortalized,often by fusion with a myeloma cell, as described in Kohler & Milstein,1975, Nature 256:495-497. For a fuller description of the production ofmonoclonal antibodies, see Antibodies: A Laboratory Manual, Harlow &Lane, eds., Cold Spring Harbor Laboratory Press, 1988.

[0065] Normal and mutant ELF proteins differ in size (normal ELF is 41amino acid longer which translates in the 4 kiloDalton difference on theSDS-PAGE). Such a difference can be easily detected, so antibodiesagainst the common parts of the two proteins can be used on Westernblots to detect the presence of the mutant ELF.

[0066] Gene Therapy

[0067] Gene therapy may be used to introduce ELF polypeptides into thecells of target organs, e.g., the photoreceptor cells, pigmentedepithelium of the retina or other parts of the retina. Nucleotidesencoding ELF polypeptides can be ligated into viral vectors whichmediate transfer of the nucleotides by infection of recipient cells.Suitable viral vectors include retrovirus, adenovirus, adeno-associatedvirus, herpes virus, vaccinia virus, and polio virus based vectors.Alternatively, nucleotides encoding ELF polypeptides can be transferredinto cells for gene therapy by non-viral techniques includingreceptor-mediated targeted transfer using ligand-nucleotide conjugates,lipofection, membrane fusion, or direct microinjection. These proceduresand variations thereof are suitable for ex vivo as well as in vivo genetherapy. Gene therapy with ELF polypeptides will be particularly usefulfor the treatment of diseases where it is beneficial to elevate ELFactivity.

[0068] Promoter/5-regulatory region of the ELF gene can be used insuitable viral and non-viral vectors to target the expression of othergenes specifically in the photoreceptor cells of the human retina, dueto the unique photoreceptor cell specificity of the ELF genetranscription. FIG. 16 shows the promoter for human ELF.

[0069] The following non-limiting Examples are presented to betterillustrate the invention.

EXAMPLE 1 Identification of the ELF Gene and cDNA Cloning Identificationof the PAC (P1 Artificial Chromosome) Clone Containing the ELF Gene

[0070] Genetics mapping clearly demonstrated the linkage of theautosomal dominant Stargardt-like macular dystrophy gene to the geneticsmarkers on human chromosome 6q14 (Edwards et al., 1999 Am. J.Ophthalmol. 127: 426-435; Griesinger et al., 2000 Inv. Ophthamol. Vis.Sci. 41: 248-255; Stone et al. 1994, Arci. Ophthalmol. 112: 765-772;each of which is incorporated by reference). The highest lod-score inthe three papers cited above was reported by Edwards et al. for thegenetic marker D6S460. The DNA sequence for D6S460 is available from thepublic DNA database (GenBank accession number Z24323).

[0071] DNA sequence from D6S460 was compared with GenBank databaseentries using the BLASTN algorithm. This comparison revealed that D6S460is contained within the DNA sequence of PAC dJ75K24 (GenBank accessionnumber AL035700).

[0072] The analysis of the physical map of human chromosome 6 availablefrom the web site of The Sanger Centre(http://www.sanger.ac.uk/HGP/Chr6/) revealed that dJ75K24 overlaps withanother PAC clone dJ159 G1 which in turn overlaps with PAC dJ92C4. Thesethree PAC clones were chosen for the detailed bioinformatic analysis.

[0073] While complete DNA sequences were available for PACs 75k24 and159G19 (GenBank accession numbers AL035700 and AL078462, respectively),the database entry for PACs 92c4 represented 11 unordered DNA piecesgenerated in Phase 1 High Throughput Genome Sequence Project (HTGS phase1)—GenBank accession number AL132875. DNA sequences of PACs 75k24,159G19, as well as the DNA sequences of 11 fragments from PAC 92c4 werecompared with GenBank database entries using the BLASTN and BLASTXalgorithms.

[0074] This comparison revealed the presence of two potential exons inPAC 92c4 whose DNA sequences, when translated, demonstrated significanthomology with the members of the yeast ELO family known to be involvedin elongation of fatty acids. Based on this homology, the novel humangene found in PAC 92s4 was designated ELF (Elongation of Fatty Acids);the two potential exons within PAC 92c4 were later defined as exons 2and 4 of the human ELF gene (see FIG. 7)

[0075] cDNA sequencing identification additional exons and exon/intronorganization of the ELF gene. The DNA sequence of the cDNA fragment thatmatches exons 2 and 4 was deduced from the genomic sequence of PAC 94c2.To identify additional exon(s) that may be located between exons 2 and4, forward and reverse PCR primers from these exons of the ELF gene weresynthesized and used to PCR amplify ELF cDNA fragments from human retina“Marathon-ready” cDNA (Clontech, Palo Alto, Calif.). In this RT-PCRexperiment forward primer from ex2 (63exDL1: GTG TGG AAA ATT GGC CTC TG)(SEQ.ID.NO. 15) was paired with a reverse primer from ex4 (63exER1: GTCCTC CTG CAA CCC AGT TA) (SEQ.ID.NO. 16). A 50 μl PCR reaction wasperformed using the Taq Gold DNA polymerase (Perkin Elmer, Norwalk,Conn.) in the reaction buffer supplied by the manufacturer with theaddition of dNTPs, primers, and approximately 0.5 ng of human retinacDNA. Cycling conditions were as follows: 1) 94° C. for 10 min; 2) 94°C. for 30 sec; 3) 72° C. for 2 min (decrease this temperature by 1.1° C.per cycle); 4) 72° C. for 2 min; 5)Go to step 2 fifteen more times; 6)94° C. for 30 sec; 7) 55° C. for 2 min; 8) 72° C. for 2 min; 9) Go tostep 6 twenty four more times; 10). 72° C. for 7 min; and 11) 4° C.

[0076] The PCR product was electrophoresed on a 2% agarose gel and DNAband was excised, purified and subjected to sequence analysis with thesame primers that were used for PCR amplification. DNA sequence analysiswas performed using the ABI PRISM™ dye terminator cycle sequencing readyreaction kit with AmpliTaq DNA polymerase, FS (Perkin Elmer, Norwalk,Conn.). Following linear amplification in a Perkin-Elmer 9600, theextension products were purified and analyzed on an ABI PRISM 377automated sequencer (Perkin Elmer, Norwalk, Conn.).

[0077] The assembly of the DNA sequence results of this PCR productrevealed that there is an additional exon between exons 2 and 4; it waslater designated exon 3. This finding defined the order of the exons inELF cDNA fragment as 5′-ex2-ex3-ex4-3′. Comparison of the DNA sequenceof exon 3 with the DNA sequence of PAC 92c4 confirmed its locationbetween exons 2 and 4 and revealed the description of intronic sequencesflanking this exon.

[0078] The DNA sequence of exon 4 was compared with the EST databaseusing the BlastN algorithm in an attempt to identify additional cDNAsequences. This analysis identified a mouse skin EST (GenBank accessionnumber AA791133) with very high degree of similarity to exon 4 of thehuman ELF gene. The DNA sequence of the mouse skin EST AA791133 wascompared with the genomic sequence of PAC 92c4. Despite the differencesbetween the mouse and human sequences caused by evolutionary divergence,this analysis was able to reveal two additional human exons with PAC94c4; there were later called exons 5 and 6. This finding defined theorder of the exons in ELF cDNA as 5′-ex2-ex3-ex4-ex5-ex6-3′.

[0079] To verify the exonic composition of the cDNA that relied at themoment on identification of exons within the genomic sequence, forwardand reverse PCR primers from known exons of the ELF gene weresynthesized and used to PCR amplify CG1CE cDNA fragments from humanretina “Marathon-ready” cDNA (Clontech, Palo Alto, Calif.). In theseRT-PCR experiments forward primer from ex2 (63exDL1: GTG TGG AAA ATT GGCCTC TG)(SEQ.ID.NO. 15) was paired with a reverse primer from ex6(63exHR1: CAT GGC TGT TTT TCC AGC TT) (SEQ.ID. NO. 17). Forward primerfrom ex5 (63exGL1: CCC AGT TGA ATT CCT TTA TCC A) (SEQ.ID.NO. 18) waspaired with a reverse primer from ex6 (63exH_Right: GTC AAC AAC AGT TAAGGC CCA) (SEQ.ID.NO. 19).

[0080] A 50 μl PCR reaction was performed using the Taq Gold DNApolymerase (Perk in Elmer, Norwalk, Con.) in the reaction buffersupplied by the manufacturer with the addition of dNTPs, primers, andapproximately 0.5 ng of human retina cDNA. PCR products wereelectrophoresed on a 2% agarose gel and DNA bands were excised, purifiedand subjected to sequence analysis with the same primers that were usedfor PCR amplification. The assembly of the DNA sequence results of thesePCR products confirmed the cDNA sequence assembled from ELF exons andcorrected the sequencing errors present in the database entry for PAC92c4.

[0081] Identification of the 5′ of the ELF cDNA

[0082] RACE is an established protocol for the analysis of cDNA ends.This procedure was performed using the Marathon RACE template from humanretina, purchased from Clontech (Palo Alto, Calif.). cDNA primer fromexon 2 (63exDR1: AGG TTA AGC AAA ACC ATC CCA) (SEQ.ID.NO. 20) incombination with a cDNA adaptor primer AP1 (CCA TCC TAA TAC GAC TCA CTATAG GGC) (SEQ.ID.NO.: 21) were used in 5′ RACE.

[0083] After the initial PCR amplification, a nested PCR reaction wasperformed using nested adaptor primer AP2 (ACT CAC TAT AGG GCT CGA GCGGC) (SEQ.ID.NO.: 22) and gene specific primer 63exDR2 (AGG TTC TCG GTCCTT CAT CC) (SEQ.ID.NO.: 23). The PCR product was separated from theunincorporated dNTP's and primers using Qiagen, QIAquick PCRpurification spin columns using standard protocols and resuspended in 30μl of water. The products were analyzed on ABI 377 sequencers accordingto standard protocols. The PCR fragment obtained in the 5′RACE reactionwas assembled into a contig with the ELF cDNA fragment covering exons 2to 6; the DNA sequence of the resulted cDNA encodes a full-length ELFprotein; the order of the exons in ELF cDNA was defined as5′-ex1-ex2-ex3-ex4-ex5-ex6-3′

[0084] Comparison of the DNA sequences obtained from RT-PCR fragmentswith genomic sequence obtained from PAC 92c4 was performed using theprogram Crossmatch. This analysis determined Exact sequence ofexon/intron boundaries within the ELF gene for all 6 exons. The splicesignals in all introns conforms to published consensus sequences.Description of the flanking intronic sequences for each of the exonsallowed the design of PCR primers for amplification of the ELF geneexons from the DNA of affected and nonaffected individuals from familieswith Stargardt-like dominant macular dystrophy.

EXAMPLE 2

[0085] Stargardt-Like Dominant Macular Dystrophy is Associated With the5-bp Deletion in the Evolutionary Conserved Region of the ELF Gene

[0086] Genomic DNA from the patients and control individuals from threepedigrees having dominant Stargardt-like macular dystrophy (families A,C, and D) was amplified by PCR using the following primer pair:63exH_Left (GAA GAT GCC GAT GTT GTT AAA AG) (SEQ.ID.NO.: 24) 63exH_Right(GTC AAC AAC AGT TAA GGC CCA) (SEQ.ID.NO. 19) This primer pair amplifiesa genomic fragment that contains exon 6 and an adjacent intronic region.

[0087] PCR products produced using the primer sets mentioned above wereamplified in 50 μl reactions consisting of Perkin-Elmer 10×PCR Buffer,200 mM dNTP's, 0.5 ul of Taq Gold (Perkin-Elmer Corp., Foster City,Calif.), 50 ng of patient DNA and 0.2 μM of forward and reverse primers.Cycling conditions were as follows: 1) 94° C. for 10 min; 2) 94° C. for30 sec; 3) 72° C. for 2 min (decrease this temperature by 1.1° C. percycle); 4) 72° C. for 2 min; 5) Go to step 2 fifteen more times; 6) 94°C. for 30 sec. 7) 55° C. for 2 min; 8) 72° C. for 2 min; 9) Go to step 6twenty four more times; 10) 72° C. for 7 min; and 11) 4° C.

[0088] Products obtained from this PCR amplification were analyzed on 2%agarose gels and excised fragments from the gels were purified usingQiagen QIAquick spin columns and sequenced using ABI dye-terminatorsequencing kits. The products were analyzed on ABI 377 sequencersaccording to standard protocols.

[0089] The results of this experiment in four individuals from family Ais shown in FIG. 12. The figure shows a small branch of this pedigreeand four sequencing runs (boxed) of PCR fragments that represent exon 6and adjacent intronic regions of the human ELF gene. From left to right,the runs are from A40 (father, unaffected with Stargardt-like dominantmacular dystrophy), A4 (mother, affected with Stargardt-like dominantmacular dystrophy), A430 (son of A4 and A40, unaffected withStargardt-like dominant macular dystrophy), A43 (daughter of A4 and A40,affected with Stargardt-like dominant macular dystrophy). Reading theboxed chromatograms from left to right, the 5-base pair deletion showsup as appearance of double peaks starting from position 7 in the case ofpatients A4 and A43. This disease mutation was not found upon sequencingof 50 normal unrelated individuals (100 chromosomes) of North Americandescent.

Example 3 Expression Studies of the ELF Gene

[0090] RT-PCR

[0091] RT-PCR experiments were performed on “quick-clone” human cDNAsamples available from Clontech, Palo Alto, Calif. ARPE-19 cDNA wasprepared according to standard protocols. cDNA samples from heart,brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, retina,testis, and human retinal pigment epithelium cell line ARPE-19 wereamplified with primers 63exGL1 (CCC AGT TGA ATT CCT TTA TCC A)(SEQ.ID.NO. 18) and 63exHR1 (CAT GGC TGT TTT TCC AGC TT) (SEQ.ID.NO. 17)in the following PCR conditions: 1) 94° C. for 10 min; 2) 94° C. for 30sec; 3) 72° C. for 2 min (decrease this temperature by 1.1° C. percycle); 4) 72° C. for 2 min; 5) Go to step 2 fifteen more times; 6) 94°C. for 30 sec; 7) 55° C. for 2 min; 8) 72° C. for 2 min; 9) Go to step 6seventeen more times; 10) 72° C. for 7 min; and 11) 4° C.

[0092] The ELF gene was found to be expressed in human retina only (FIG.14).

[0093] Northern Blot Analysis

[0094] Northern blots containing poly(A+)-RNA from different humantissues were purchased from Clontech, Palo Alto, Calif. The blotcontained human heart, brain placenta, lung, liver, skeletal muscle,kidney, and pancreas poly(A+)-RNA. A custom-made blot containing humanretina, brain, and ARPE-19 poly(A⁺)-RNA was ordered from FRP Grating.Primers 63exDL1 (GTG TGG AAA ATT GGC CTC TG) (SEQ.ID.NO. 15) and 63exHR1(CAT GGC TGT TTT TCC AGC TT) (SEQ.ID.NO. 17) were used to amplify a PCRproduct from the “quick-clone” human retina cDNA available fromClontech, Palo Alto, Calif. This product was purified on an agarose gel,and used as a probe in Northern blot hybridization. The probe waslabeled by random priming with the Amersham Rediprime kit (ArlingtonHeights, Ill.) in the presence of 50-100 μCi of 3000 Ci/mmole [alpha³²P]dCTP (Dupont/NEN, Boston, Mass.). Unincorporated nucleotides wereremoved with a ProbeQuant G-50 spin column (Phannacia/Biotech,Piscataway, N.J.). The radiolabeled probe at a concentration of greaterthan 1×10⁶ cpm/ml in rapid hybridization buffer (Clontech, Palo Alto,Calif.) was incubated overnight at 65° C. The blots were washed by two15 min incubations in 2×SSC, 0.1% SDS (prepared from 20×SSC and 20% SDSstock solutions, Fisher, Pittsburgh, Pa.) at room temperature, followedby two 15 min incubations in 1×SSC, 0.1% SDS at room temperature, andtwo 30 min incubations in 0.1×SSC, 0.1% SDS at 60° C. Autoradiography ofthe blots was done to visualize the bands that specifically hybridizedto the radiolabeled probe.

[0095] The probe hybridized to an mRNA transcript that is uniquelyexpressed in the human retina (see FIG. 15). Weaker hybridization signalis also seen in the human brain. ELF mRNA exists in two differentspecies, similar to what was reported for its only mammal relative, theCig30 gene (Tvrdik et al., J. Biol. Chem., 1999, 274:26387-26392; whichis hereby incorporated by reference).

[0096] In situ hybridization

[0097] Primers 63exGL1 (CCC AGT TGA ATT CCT TTA TCC A) (SEQ.ID.NO. 18)and 63exHR1 (CAT GGC TGT TTT TCC AGC TT) (SEQ.ID.NO. 17) were used toamplify a PCR product from the “quick-clone” human retina cDNA availablefrom Clontech, Palo Alto, Calif. This product was subcloned into thepCR-Script vector (Stratagene) giving the plasmid called phumGL1/HR2.This plasmid served as a hybridization probe and represented a fragmentof the human normal ELF cDNA with coordinates 561-771. In situhybridization was carried out on sections of rhesus monkey retinaaccording to standard protocols. Specific expression is seen in theinner segments of photoreceptor cells with the antisense probe (leftpanel). Probe signal is seen in blue color; retinal layers arevisualized with propidium iodide counterstain (red). Right panel showsthe hybridization with the sense control probe (sense probe is notcomplementary to the ELF mRNA). Retinal layers are marked as RPE,retinal pigment epithelium; OS, outer segments of photoreceptors; IS,inner segments of photoreceptors; ONL, outer nuclear layer; OPL, outerplexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer;GCL, ganglion cell layer.

1 24 1 314 PRT Human 1 Met Gly Leu Leu Asp Ser Glu Pro Gly Ser Val LeuAsn Val Val Ser 1 5 10 15 Thr Ala Leu Asn Asp Thr Val Glu Phe Tyr ArgTrp Thr Trp Ser Ile 20 25 30 Ala Asp Lys Arg Val Glu Asn Trp Pro Leu MetGln Ser Pro Trp Pro 35 40 45 Thr Leu Ser Ile Ser Thr Leu Tyr Leu Leu PheVal Trp Leu Gly Pro 50 55 60 Lys Trp Met Lys Asp Arg Glu Pro Phe Gln MetArg Leu Val Leu Ile 65 70 75 80 Ile Tyr Asn Phe Gly Met Val Leu Leu AsnLeu Phe Ile Phe Arg Glu 85 90 95 Leu Phe Met Gly Ser Tyr Asn Ala Gly TyrSer Tyr Ile Cys Gln Ser 100 105 110 Val Asp Tyr Ser Asn Asn Val His GluVal Arg Ile Ala Ala Ala Leu 115 120 125 Trp Trp Tyr Phe Val Ser Lys GlyVal Glu Tyr Leu Asp Thr Val Phe 130 135 140 Phe Ile Leu Arg Lys Lys AsnAsn Gln Val Ser Phe Leu His Val Tyr 145 150 155 160 His His Cys Thr MetPhe Thr Leu Trp Trp Ile Gly Ile Lys Trp Val 165 170 175 Ala Gly Gly GlnAla Phe Phe Gly Ala Gln Leu Asn Ser Phe Ile His 180 185 190 Val Ile MetTyr Ser Tyr Tyr Gly Leu Thr Ala Phe Gly Pro Trp Ile 195 200 205 Gln LysTyr Leu Trp Trp Lys Arg Tyr Leu Thr Met Leu Gln Leu Ile 210 215 220 GlnPhe His Val Thr Ile Gly His Thr Ala Leu Ser Leu Tyr Thr Asp 225 230 235240 Cys Pro Phe Pro Lys Trp Met His Trp Ala Leu Ile Ala Tyr Ala Ile 245250 255 Ser Phe Ile Phe Leu Phe Leu Asn Phe Tyr Ile Arg Thr Tyr Lys Glu260 265 270 Pro Lys Lys Pro Lys Ala Gly Lys Thr Ala Met Asn Gly Ile SerAla 275 280 285 Asn Gly Val Ser Lys Ser Glu Lys Gln Leu Met Ile Glu AsnGly Lys 290 295 300 Lys Gln Lys Asn Gly Lys Ala Lys Gly Asp 305 310 2271 PRT Human 2 Met Gly Leu Leu Asp Ser Glu Pro Gly Ser Val Leu Asn ValVal Ser 1 5 10 15 Thr Ala Leu Asn Asp Thr Val Glu Phe Tyr Arg Trp ThrTrp Ser Ile 20 25 30 Ala Asp Lys Arg Val Glu Asn Trp Pro Leu Met Gln SerPro Trp Pro 35 40 45 Thr Leu Ser Ile Ser Thr Leu Tyr Leu Leu Phe Val TrpLeu Gly Pro 50 55 60 Lys Trp Met Lys Asp Arg Glu Pro Phe Gln Met Arg LeuVal Leu Ile 65 70 75 80 Ile Tyr Asn Phe Gly Met Val Leu Leu Asn Leu PheIle Phe Arg Glu 85 90 95 Leu Phe Met Gly Ser Tyr Asn Ala Gly Tyr Ser TyrIle Cys Gln Ser 100 105 110 Val Asp Tyr Ser Asn Asn Val His Glu Val ArgIle Ala Ala Ala Leu 115 120 125 Trp Trp Tyr Phe Val Ser Lys Gly Val GluTyr Leu Asp Thr Val Phe 130 135 140 Phe Ile Leu Arg Lys Lys Asn Asn GlnVal Ser Phe Leu His Val Tyr 145 150 155 160 His His Cys Thr Met Phe ThrLeu Trp Trp Ile Gly Ile Lys Trp Val 165 170 175 Ala Gly Gly Gln Ala PhePhe Gly Ala Gln Leu Asn Ser Phe Ile His 180 185 190 Val Ile Met Tyr SerTyr Tyr Gly Leu Thr Ala Phe Gly Pro Trp Ile 195 200 205 Gln Lys Tyr LeuTrp Trp Lys Arg Tyr Leu Thr Met Leu Gln Leu Ile 210 215 220 Gln Phe HisVal Thr Ile Gly His Thr Ala Leu Ser Leu Tyr Thr Asp 225 230 235 240 CysPro Phe Pro Lys Trp Met His Trp Ala Leu Ile Ala Tyr Ala Ile 245 250 255Ser Phe Ile Phe Leu Phe Leu Leu His Ser Asp Ile Gln Arg Ala 260 265 2703 972 DNA Human 3 cgccgcgatg gggctcctgg actcggagcc gggtagtgtc ctaaacgtagtgtccacggc 60 actcaacgac acggtagagt tctaccgctg gacctggtcc atcgcagataagcgtgtgga 120 aaattggcct ctgatgcagt ctccttggcc tacactaagt ataagcactctttatctcct 180 gtttgtgtgg ctgggtccaa aatggatgaa ggaccgagaa ccttttcagatgcgtctagt 240 gctcattatc tataattttg ggatggtttt gcttaacctc tttatcttcagagagttatt 300 catgggatca tataatgcgg gatatagcta tatttgccag agtgtggattattctaataa 360 tgttcatgaa gtcaggatag ctgctgctct gtggtggtac tttgtatctaaaggagttga 420 gtatttggac acagtgtttt ttattctgag aaagaaaaac aaccaagtttctttccttca 480 tgtgtatcat cactgtacga tgtttacctt gtggtggatt ggaattaagtgggttgcagg 540 aggacaagca ttttttggag cccagttgaa ttcctttatc catgtgattatgtactcata 600 ctatgggtta actgcatttg gcccatggat tcagaaatat ctttggtggaaacgatacct 660 gactatgttg caactgattc aattccatgt gaccattggg cacacggcactgtctcttta 720 cactgactgc cccttcccca aatggatgca ctgggctcta attgcctatgcaatcagctt 780 catatttctc tttcttaact tctacattcg gacatacaaa gagcctaagaaaccaaaagc 840 tggaaaaaca gccatgaatg gtatttcagc aaatggtgtg agcaaatcagaaaaacaact 900 catgatagaa aatggaaaaa agcagaaaaa tggaaaagca aaaggagattaaattgaact 960 gggccttaac tg 972 4 967 DNA Human 4 cgccgcgatg gggctcctggactcggagcc gggtagtgtc ctaaacgtag tgtccacggc 60 actcaacgac acggtagagttctaccgctg gacctggtcc atcgcagata agcgtgtgga 120 aaattggcct ctgatgcagtctccttggcc tacactaagt ataagcactc tttatctcct 180 gtttgtgtgg ctgggtccaaaatggatgaa ggaccgagaa ccttttcaga tgcgtctagt 240 gctcattatc tataattttgggatggtttt gcttaacctc tttatcttca gagagttatt 300 catgggatca tataatgcgggatatagcta tatttgccag agtgtggatt attctaataa 360 tgttcatgaa gtcaggatagctgctgctct gtggtggtac tttgtatcta aaggagttga 420 gtatttggac acagtgttttttattctgag aaagaaaaac aaccaagttt ctttccttca 480 tgtgtatcat cactgtacgatgtttacctt gtggtggatt ggaattaagt gggttgcagg 540 aggacaagca ttttttggagcccagttgaa ttcctttatc catgtgatta tgtactcata 600 ctatgggtta actgcatttggcccatggat tcagaaatat ctttggtgga aacgatacct 660 gactatgttg caactgattcaattccatgt gaccattggg cacacggcac tgtctcttta 720 cactgactgc cccttccccaaatggatgca ctgggctcta attgcctatg caatcagctt 780 catatttctc tttcttctacattcggacat acaaagagcc taagaaacca aaagctggaa 840 aaacagccat gaatggtatttcagcaaatg gtgtgagcaa atcagaaaaa caactcatga 900 tagaaaatgg aaaaaagcagaaaaatggaa aagcaaaagg agattaaatt gaactgggcc 960 ttaactg 967 5 312 PRTMus Musculus 5 Met Gly Leu Leu Asp Ser Glu Pro Gly Ser Val Leu Asn AlaMet Ser 1 5 10 15 Thr Ala Phe Asn Asp Thr Val Glu Phe Tyr Arg Trp ThrTrp Thr Ile 20 25 30 Ala Asp Lys Arg Val Ala Asp Trp Pro Leu Met Gln SerPro Trp Pro 35 40 45 Thr Ile Ser Ile Ser Thr Leu Tyr Leu Leu Phe Val TrpLeu Gly Pro 50 55 60 Lys Trp Met Lys Asp Arg Glu Pro Phe Gln Met Arg LeuVal Leu Ile 65 70 75 80 Ile Tyr Asn Phe Gly Met Val Leu Leu Asn Leu PheIle Phe Arg Glu 85 90 95 Leu Phe Met Gly Ser Tyr Asn Ala Gly Tyr Ser TyrIle Cys Gln Ser 100 105 110 Val Asp Tyr Ser Asn Asp Val Asn Glu Val ArgIle Ala Ala Ala Leu 115 120 125 Trp Trp Tyr Phe Val Ser Lys Gly Val GluTyr Leu Asp Thr Val Phe 130 135 140 Phe Ile Leu Arg Lys Lys Asn Asn GlnVal Ser Phe Leu His Val Tyr 145 150 155 160 His His Cys Thr Met Phe ThrLeu Trp Trp Ile Gly Ile Lys Trp Val 165 170 175 Ala Gly Gly Gln Ala PhePhe Gly Ala Gln Met Asn Ser Phe Ile His 180 185 190 Val Ile Met Tyr SerTyr Tyr Gly Leu Thr Ala Phe Gly Pro Trp Ile 195 200 205 Gln Lys Tyr LeuTrp Trp Lys Arg Tyr Leu Thr Met Leu Gln Leu Val 210 215 220 Gln Phe HisVal Thr Ile Gly His Thr Ala Leu Ser Leu Tyr Thr Asp 225 230 235 240 CysPro Phe Pro Lys Trp Met His Trp Ala Leu Ile Ala Tyr Ala Ile 245 250 255Ser Phe Ile Phe Leu Phe Leu Asn Phe Tyr Thr Arg Thr Tyr Asn Glu 260 265270 Pro Lys Gln Ser Lys Thr Gly Lys Thr Ala Thr Asn Gly Ile Ser Ser 275280 285 Asn Gly Val Asn Lys Ser Glu Lys Ala Leu Glu Asn Gly Lys Pro Gln290 295 300 Lys Asn Gly Lys Pro Lys Gly Glu 305 310 6 1292 DNA MusMusculus 6 cagtcgccca cggtccatcg gagcctctct tctcgcccgc ttgtcgtacctctcctcgcc 60 aagatggggc tgctggactc agagcccggc agcgtcctga acgcgatgtccacggcattc 120 aacgacaccg tggagttcta tcgctggacc tggaccattg cagataaacgtgtagcagac 180 tggccgctga tgcagtctcc atggccaacg ataagcataa gcacgctctatctcctgttc 240 gtgtggctgg gtccaaagtg gatgaaagac cgcgagccgt tccaaatgcgcttagtactc 300 ataatctata attttggcat ggttttgctt aaccttttca tcttcagagagctattcatg 360 ggatcataca acgcaggata cagctatatt tgccagtcag tggattattctaatgatgtt 420 aatgaagtca ggatagcggc ggccctgtgg tggtattttg tatcgaaaggcgttgagtat 480 ttggacacag tgttttttat cctgaggaag aaaaacaacc aagtctccttccttcacgtg 540 taccaccact gcaccatgtt cactctgtgg tggattggaa tcaagtgggtggctggaggc 600 caagcgtttt tcggggccca gatgaactct ttcatccacg tgatcatgtactcctactat 660 gggctgactg cgttcggccc ctggatccag aaatatcttt ggtggaagcgatacctgacc 720 atgctgcagc tggtccagtt ccacgtgacc atcggacaca cagcactgtctctctacacc 780 gactgcccct tccccaagtg gatgcactgg gctctgatcg cctacgccatcagcttcatc 840 ttcctcttcc tcaacttcta cactcggaca tacaatgagc cgaagcagtcaaaaaccgga 900 aagacggcca cgaatggtat ctcatcgaac ggcgtgaata aatcagagaaagcgttagaa 960 aacgggaaac cccagaaaaa cgggaagcca aaaggagagt aaattgaactgggccttaac 1020 cggtagacag tgaggaaact cctgtgtcat tttaaaaagt tcaggggcaacagaagcaga 1080 gggtctgggc tggggagaaa ggcagatagg gtctttgccc ttcagactgagtaaaacttt 1140 tcaatatatg gtacccagat gttttattta tgaagttttt attttaaaagtttttttttt 1200 attaaccctt catgttgtcc aaaaccaaag caacccccaa tgtggaccttgggagccttt 1260 tctctgttaa cattccgcct tgggcaatgg gg 1292 7 2395 DNAHuman Variable length; some residues may be missing 7 gccgccaccgcctccggggt cagccctctc tctgggtctc cgctttctcc tgccgccagc 60 gcccgctcatcgccgcgatg gggctcctgg actcggagcc gggtagtgtc ctaaacgtag 120 tgtccacggcactcaacgac acggtagagt tctaccgctg gacctggtcc atcgcaggta 180 aagccgctgacttccccatc ctcgctcggt cccccgcggg gggtcaccgg cccctggtct 240 cgcagctcccgggcccggcc ccacaggccc ccgcgccctg cggctttcgg atgctgcgga 300 agccacttgcaggagtcagt attgtttctt tggtttttat accatgtatt ttttgttggg 360 actcaaaggacagtgatccg tatttagtca aattaggaaa ttaagttgaa acatcttgat 420 tcctaaaaagtgtattttat aaaacattta ctgattaatg aattttatgg tattttgttc 480 tctctatagataagcgtgtg gaaaattggc ctctgatgca gtctccttgg cctacactaa 540 gtataagcactctttatctc ctgtttgtgt ggctgggtcc aaaatggatg aaggaccgag 600 aaccttttcagatgcgtcta gtgctcatta tctataattt tgggatggtt ttgcttaacc 660 tctttatcttcagagaggta tgtttttaag atcactttaa taattttcca aggttattgg 720 aaatttaaaaatgagaatgt gtaaaaccat aatcggaatg catgaaattt ttaatgcatt 780 tgaaatttttaaagaaaata ttgtgtttaa aataatttga aaggctacat tttgtatata 840 attgtgtttttaatgctgtg tttactaaaa ctttactaca aatattatta ctctttttcc 900 agttattcatgggatcatat aatgcgggat atagctatat ttgccagagt gtggattatt 960 ctaataatgttcatgaagtc agggtaagta cattaaaaat actcttaatc agtaaaagtg 1020 gtttgatttttataggcccc agtctgtgaa aatccatgcc ttgtacattt tgtgcaatat 1080 acaaatgtttattttggast tacttacaat gagtataaac ccatacaata gtgtcatttt 1140 ggtgtttataacacgctttc cctttttaca gatagctgct gctctgtggt ggtactttgt 1200 atctaaaggagttgagtatt tggacacagt gttttttatt ctgagaaaga aaaacaacca 1260 agtttctttccttcatgtgt atcatcactg tacgatgttt accttgtggt ggattggaat 1320 taagtgggttgcaggaggac aaggtgagca ttttcaggaa tatactgctt gcgtttaatt 1380 gcatatatgtgttcagtgga aagcaatgag aacctaggac tttgacttga tctaccattt 1440 aacttgctttcatggttaat catttccatg ttcatttctt tttttttttt tttttttttt 1500 ttttgagatggagtctcgct ctgtcaccag gctggagtgc agtggcgcga tctcggctca 1560 ctgcaacctccacctcccgg gttccagcga ttctcctgcc tcagcctcct gagtagctgg 1620 gactacaggcacacaccacc acgcctagct aattttttgt atttttagta gagacagggt 1680 ttcaccatgttggccaggat ggtaaaagat ctcttgacct tgtgatccgc catctcagtg 1740 gcttactgcctaataaaatt ttctgtatct tgtaattacc tgttgttttt ctaaagcatt 1800 ttttggagcccagttgaatt cctttatcca tgtgattatg tactcatact atgggttaac 1860 tgcatttggcccatggattc agaaatatct ttggtggaaa cgatacctga ctatgttgca 1920 actggtgagttaaatgcttc caaagtttct tctggtaaaa tactgaaatt gtttaaattt 1980 gattaattttaaagtgcaat gtcattttag acaattttca gatgccgatg ttgttaaaag 2040 ttgtttactattcagattaa atgttttgtg ctgtcatttc tgtttttcag attcaattcc 2100 atgtgaccattgggcacacg gcactgtctc tttacactga ctgccccttc cccaaatgga 2160 tgcactgggctctaattgcc tatgcaatca gcttcatatt tctctttctt aacttctaca 2220 ttcggacatacaaagagcct aagaaaccaa aagctggaaa aacagccatg aatggtattt 2280 cagcaaatggtgtgagcaaa tcagaaaaac aactcatgat agaaaatgga aaaaagcaga 2340 aaaatggaaaagcaaaagga gattaaattg aactgggcct taactgttgt tgaca 2395 8 347 PRTSaccharomyces 8 Met Asn Ser Leu Val Thr Gln Tyr Ala Ala Pro Leu Phe GluArg Tyr 1 5 10 15 Pro Gln Leu His Asp Tyr Leu Pro Thr Leu Glu Arg ProPhe Phe Asn 20 25 30 Ile Ser Leu Trp Glu His Phe Asp Asp Val Val Thr ArgVal Thr Asn 35 40 45 Gly Arg Phe Val Pro Ser Glu Phe Gln Phe Ile Ala GlyGlu Leu Pro 50 55 60 Leu Ser Thr Leu Pro Pro Val Leu Tyr Ala Ile Thr AlaTyr Tyr Val 65 70 75 80 Ile Ile Phe Gly Gly Arg Phe Leu Leu Ser Lys SerLys Pro Phe Lys 85 90 95 Leu Asn Gly Leu Phe Gln Leu His Asn Leu Val LeuThr Ser Leu Ser 100 105 110 Leu Thr Leu Leu Leu Leu Met Val Glu Gln LeuVal Pro Ile Ile Val 115 120 125 Gln His Gly Leu Tyr Phe Ala Ile Cys AsnIle Gly Ala Trp Thr Gln 130 135 140 Pro Leu Val Thr Leu Tyr Tyr Met AsnTyr Ile Val Lys Phe Ile Glu 145 150 155 160 Phe Ile Asp Thr Phe Phe LeuVal Leu Lys His Lys Lys Leu Thr Phe 165 170 175 Leu His Thr Tyr His HisGly Ala Thr Ala Leu Leu Cys Tyr Thr Gln 180 185 190 Leu Met Gly Thr ThrSer Ile Ser Trp Val Pro Ile Ser Leu Asn Leu 195 200 205 Gly Val His ValVal Met Tyr Trp Tyr Tyr Phe Leu Ala Ala Arg Gly 210 215 220 Ile Arg ValTrp Trp Lys Glu Trp Val Thr Arg Phe Gln Ile Ile Gln 225 230 235 240 PheVal Leu Asp Ile Gly Phe Ile Tyr Phe Ala Val Tyr Gln Lys Ala 245 250 255Val His Leu Tyr Phe Pro Ile Leu Pro His Cys Gly Asp Cys Val Gly 260 265270 Ser Thr Thr Ala Thr Phe Ala Gly Cys Ala Ile Ile Ser Ser Tyr Leu 275280 285 Val Leu Phe Ile Ser Phe Tyr Ile Asn Val Tyr Lys Arg Lys Gly Thr290 295 300 Lys Thr Ser Arg Val Val Lys Arg Ala His Gly Gly Val Ala AlaLys 305 310 315 320 Val Asn Glu Tyr Val Asn Val Asp Leu Lys Asn Val ProThr Pro Ser 325 330 335 Pro Ser Pro Lys Pro Gln His Arg Arg Lys Arg 340345 9 345 PRT Saccharomyces 9 Met Asn Thr Thr Thr Ser Thr Val Ile AlaAla Val Ala Asp Gln Phe 1 5 10 15 Gln Ser Leu Asn Ser Ser Ser Ser CysPhe Leu Lys Val His Val Pro 20 25 30 Ser Ile Glu Asn Pro Phe Gly Ile GluLeu Trp Pro Ile Phe Ser Lys 35 40 45 Val Phe Glu Tyr Phe Ser Gly Tyr ProAla Glu Gln Phe Glu Phe Ile 50 55 60 His Asn Lys Thr Phe Leu Ala Asn GlyTyr His Ala Val Ser Ile Ile 65 70 75 80 Ile Val Tyr Tyr Ile Ile Ile PheGly Gly Gln Ala Ile Leu Arg Ala 85 90 95 Leu Asn Ala Ser Pro Leu Lys PheLys Leu Leu Phe Glu Ile His Asn 100 105 110 Leu Phe Leu Thr Ser Ile SerLeu Val Leu Trp Leu Leu Met Leu Glu 115 120 125 Gln Leu Val Pro Met ValTyr His Asn Gly Leu Phe Trp Ser Ile Cys 130 135 140 Ser Lys Glu Ala PheAla Pro Lys Leu Val Thr Leu Tyr Tyr Leu Asn 145 150 155 160 Tyr Leu ThrLys Phe Val Glu Leu Ile Asp Thr Val Phe Leu Val Leu 165 170 175 Arg ArgLys Lys Leu Leu Phe Leu His Thr Tyr His His Gly Ala Thr 180 185 190 AlaLeu Leu Cys Tyr Thr Gln Leu Ile Gly Arg Thr Ser Val Glu Trp 195 200 205Val Val Ile Leu Leu Asn Leu Gly Val His Val Ile Met Tyr Trp Tyr 210 215220 Tyr Phe Leu Ser Ser Cys Gly Ile Arg Val Trp Trp Lys Gln Trp Val 225230 235 240 Thr Arg Phe Gln Ile Ile Gln Phe Leu Ile Asp Leu Val Phe ValTyr 245 250 255 Phe Ala Thr Tyr Thr Phe Tyr Ala His Lys Tyr Leu Asp GlyIle Leu 260 265 270 Pro Asn Lys Gly Thr Cys Tyr Gly Thr Gln Ala Ala AlaAla Tyr Gly 275 280 285 Tyr Leu Ile Leu Thr Ser Tyr Leu Leu Leu Phe IleSer Phe Tyr Ile 290 295 300 Gln Ser Tyr Lys Lys Gly Gly Lys Lys Thr ValLys Lys Glu Ser Glu 305 310 315 320 Val Ser Gly Ser Val Ala Ser Gly SerSer Thr Gly Val Lys Thr Ser 325 330 335 Asn Thr Lys Val Ser Ser Arg LysAla 340 345 10 16 DNA Human 10 tttcttaact tctaga 16 11 16 DNA Humanmisc_feature (1)...(16) n = A,T,C or G 11 tttcttannc attncn 16 12 16 DNAHuman 12 tttcttaact tctaca 16 13 16 DNA Human misc_feature (1)...(16) n= A,T,C or G 13 tttcttanac attcgg 16 14 15 DNA Artificial Sequence Probe14 ctttcttcta cattc 15 15 20 DNA Artificial Sequence PCR Probe 15gtgtggaaaa ttggcctctg 20 16 20 DNA Artificial Sequence PCR Probe 16gtcctcctgc aacccagtta 20 17 20 DNA Artificial Sequence PCR Probe 17catggctgtt tttccagctt 20 18 22 DNA Artificial Sequence PCR Probe 18cccagttgaa ttcctttatc ca 22 19 21 DNA Artificial Sequence PCR Probe 19gtcaacaaca gttaaggccc a 21 20 21 DNA Artificial Sequence PCR Probe 20aggttaagca aaaccatccc a 21 21 27 DNA Artificial Sequence PCR Probe 21ccatcctaat acgactcact atagggc 27 22 23 DNA Artificial Sequence PCR Probe22 actcactata gggctcgagc ggc 23 23 20 DNA Artificial Sequence PCR Probe23 aggttctcgg tccttcatcc 20 24 23 DNA Artificial Sequence PCR Probe 24gaagatgccg atgttgttaa aag 23

What is claimed is:
 1. An Elongation of Fatty Acids (ELF) protein, freefrom associated proteins, comprising the amino acids shown inSEQ.ID.NO.
 1. 2. A pharmaceutical composition comprising the protein ofclaim 1 and a pharmaceutically acceptable carrier.
 3. A pharmaceuticalcomposition according to claim 2 wherein the composition is anophthalmic composition.
 4. A method of treating, preventing or lesseningthe severity of Stargardt-like macular dystrophy comprisingadministering a pharmacologically effective amount of the composition ofclaim 2 to an individual at risk or who manifest symptoms.
 5. AnElongation of Fatty Acids (ELF) protein, free from associated proteins,comprising the amino acids shown in SEQ.ID.NO.
 2. 6. Mouse ELF protein,free from associated protein, comprising the amino acids shown inSEQ.ID.NO.
 5. 7. A method of determining if an individual is at risk fordeveloping symptoms of Stargardt-like macular dystrophy comprisingdetermining if the individual carries a gene encoding the protein ofSEQ.ID.NO. 1 or SEQ.ID.NO. 2 wherein the protein of SEQ.ID.NO. 2 isassociated with Stargardt-like macular dystrophy, and the protein ofSEQ.ID.NO. 1 is not.
 8. A method according to claim 7 wherein thedetermination is an assay comprising contacting a probe and a nucleicacid sample of the individual, wherein hybridization of the probe andthe nucleic acids in the sample indicates that a normal ELF nucleic acidis present in the sample.
 9. A method according to claim 7 wherein thedetermination is an assay comprising contacting a probe and a nucleicacid sample of the individual, wherein hybridization of the probe andthe nucleic acids in the sample indicates that a mutant ELF nucleic acidis present in the sample.
 10. A method according to claim 7 wherein thedetermination is an assay comprising obtaining a sample of the proteinproduced by the individual and determining if the sample is the proteinof SEQ.ID.NO. 1 or SEQ.ID.NO.
 2. 11. A method according to claim 10wherein the determining step comprises contacting the protein samplewith an antibody specific for either the protein of SEQ.ID.NO. 1 orSEQ.ID.NO. 2, and determining if binding occurs.
 12. A method ofisolating nucleic acids encoding an ELF protein in a non-human mammalcomprising contacting a retinal library from the non-human mammal with aprobe from human ELF DNA.
 13. A nucleic acid encoding an amino acidselected from the group consisting of: SEQ.ID.NO. 1, SEQ.ID.NO. 2, andSEQ.ID.NO. 5, free from associated nucleic acids.
 14. A nucleic acid ofclaim 13 which is DNA.
 15. cDNA selected from the group consisting ofSEQ.ID.NO. 3, SEQ.ID.NO. 4, and SEQ.ID.NO.
 6. 16. A DNA, free fromassociated nucleic acids comprising the sequence of SEQ.ID.NO.
 7. 17. Avector comprising an ELF expression cassette.
 18. A host cell comprisinga vector of claim
 17. 19. A method of making an ELF protein comprisingculturing a host cell comprising an ELF expression cassette, andrecovering ELF protein.